4 PLENARY LECTURES Plenary lecture 1. Dendritic computation Michael Hausser Wolfson Institute for Biomedical Research, University College London, UK Plenary lecture 2. The earliest cortical circuits Zoltán Molnár, Anna Hoerder-Suabedissen, Franziska Oeschger, Wei Zhi Wang Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford, United Kingdom The subplate zone is a highly dynamic transient sector of the developing cerebral cortex that contains some of the earliest generated neurons, and the first functional synapses of the cerebral cortex. Subplate cells have important functions in early establishment and maturation of thalamocortical connections, as well as in the development of inhibitory cortical circuits in sensory areas. So far no role has been identified for cells in the subplate in the mature brain and disease association of the subplatespecific genes has not been analyzed systematically. Here we present gene expression evidence for distinct roles of the subplate across development as well as novel molecular markers to extend the repertoire of subplate labels. Performing systematic comparisons between different ages (E15, E18, P8 and adult) we reveal the dynamic and constant features of the novel markers labeling subplate cells during embryonic and early postnatal development and in the adult. This can be visualized using the online database of subplate gene expression at We also 4

5 identify embryonic similarities in gene expression between the ventricular zones, intermediate zone and subplate, and distinct postnatal similarities between subplate, layer 5 and layer 2/3. The genes expressed in a subplatespecific manner at some point during development show a statistically significant enrichment for association with autism spectrum disorders and schizophrenia. Our report emphasizes the importance of the study of transient features of the developing brain to better understand neurodevelopmental disorders. Plenary lecture 3. Neural syntax: organization of cell assembly sequences by brain rhythms György Buzsáki NYU Neuroscience Institute, New York University, Langone Medical Center, New York, USA Plenary lecture 4. From the nucleus to the synapse and outside in learning and memory: From c-fos to MMP-9 Leszek Kaczmarek Nencki Institute of Experimental Biology, Warsaw, Poland c-fos, a constituent of transcriptional regulator AP-1 was discovered in late eighties as driven by neuronal activity and then has been associated with plasticity, learning and memory. Next, we have identified 5

6 extracellular proteolytic system, composed of matrix metalloproteinase-9 (MMP-9) and its endogenous inhibitor, TIMP-1 (tissue inhibitor of matrix metalloproteinases) as being controlled by c-fos/ap-1 in activated neurons. Over the last decade we have extensively investigated the role of MMP-9 in the learning and memory and underlying synaptic plasticity. In brief, we have found that MMP-9 plays a role in a contextual fear conditioning as well as in appetitive, but not aversive learning in the Intellicages. Furthermore, we have shown that MMP-9 specifically in the central amygdala is critical for the reward learning. We have also shown that MMP-9 is mandatory for the late phase of long-term potentiation (LTP) in the hippocampus, prefrontal cortex and central amygdala (but not in the lateral amygdala). Furthermore, we have localized MMP-9 mrna, protein and enzymatic activity at the dendrites and dendritic spines, harboring excitatory synapses (but not at the inhibitory synapses). Locally available mrna can be translated in response to enhanced synaptic activity. We have also shown that MMP-9 controls morphological plasticity of the dendritic spines and associated mobility of glutamate receptors mobility. In aggregate, we have shown that MMP-9 is important for synaptic plasticity, learning and memory, and its understanding may explain c-fos involvement in those phenomena. Plenary lecture 5. Fejezetek az idegtudomány történetéből Miklós Palkovits Laboratory of Neuromorphology, Department of Anatomy, Histology and Embryology and the Human Brain Tissue Bank, Semmelweis University, Budapest, Hungary 6

7 SYMPOSIUM LECTURES Symposium 1. Wiring principles of the cerebral cortex S1.1. Unravelling the Neuronal Knitting Julian Budd Department of Informatics, Sussex University, Brighton, United Kingdom Mapping how neurons in cerebral cortex are interconnected the cortical connectome is widely expected to provide a foundation for explaining fundamental cognitive and perceptual functions. Yet the task of reconstructing the connectivity of even a single axon, for example, is a complicated and time consuming business. The cortical neuropil is composed of the dense interweaving of very many glial, neuronal, and vascular processes. Hence the trajectory of an axon and its branches must be carefully traced through a maze of cellular processes to identify its likely postsynaptic targets. When the vast number of distinct axons contained within even a modest volume of cortical neuropil is considered, the task of completely mapping axonal connectivity takes on an epic stature. However, a number of recent technical advances now offer the potential for much more mapping data at multiple spatial scales of cortical organization. Here I will consider the advantages and limitations of these recent advances towards achieving the goal of mapping the cortical connectome. The main focus of the talk will be to argue for the importance of discovering the nature and purpose of cortical wiring principles underlying the organization of cerebral cortex. 7

8 S1.2. In vivo whole-cell recording, transfection and transsynaptic tracing in mouse neocortex Ede Rancz, Charly Rousseau, Mateo Velez-Fort, Molly Strom, Christian Niedworok, Troy W. Margrie Neurophysiology, MRC National Institute for Medical Research, London, United Kingdom Relating structure and function is required to better understand the nervous system. To this end, several microscopy and tracing techniques have been devised and perfected to assess local and long-range connectivity in simple and more complex model organisms. I will describe a method that may be used to genetically label the presynaptic partners of a single, functionally characterized neuron. We combined in vivo whole-cell recording of synaptic and spiking receptive fields of mouse visual cortical neurons with delivering plasmids through the patch pipette. A high proportion of recorded cells survived for days and expressed exogenous proteins coded by several plasmids in the recording solution. By delivering plasmids coding for a fluorescent protein, TVA-receptor and rabies-virus glycoprotein, we made single, recorded cells exclusively susceptible to modified rabies virus infection. The modified rabies virus, expressing a fluorescent protein but lacking the glycoprotein from its genome, was then able to spread to monosynaptically coupled presynaptic partners but no further. This method thus allows us to ascertain the synaptic and anatomical receptive field of single neurons however results in large volumes of data, the acquisition and processing of which poses new challenges. I will describe some recent advances we have made on this front. S1.3. 8

9 The Blue Brain Project: In silico Neuroscience an integrative approach Felix Schürmann Blue Brain Project, EPFL, Lausanne, Switzerland Many areas of science and engineering have adopted simulation-based research as a novel tool for discovery and insight. The Blue Brain Project has pioneered this approach for brain research. On the one hand, the heterogeneity of neural systems poses particular challenges: the data is multi-modal, multi-scale and often times incomplete, intricate workflows are required for model generation, mathematical formulations are volatile, and memory and compute requirements to cope with the heterogeneity are demanding. On the other hand, neurobiology has potentially a lot to gain: systematically accounting for the data and bringing it together in a unifying computer model provides an integration strategy capable of overcoming the fragmentation of data and identifying gaps in our knowledge. Attempting this ultimate integration is revealing novel design principles of the brain. These principles are in turn helping to predictively fill gaps in data and knowledge.this approach differs from computational neuroscience in that the models are zero tweak models - the brain is built bottom up from first principles and validated top down by comparing models with biological emergent properties.as a proof of concept, the Blue Brain Project built a facility comprised of many key technologies and workflows and used this facility to build and simulate the neocortical column of the somatosensory cortex of the young rat. S1.4. Connectome and hodology, structure and function Ervin Berényi, András Jakab 9

10 Department of BioMedical Laboratory and Imaging Science, Debrecen University, Debrecen, Hungary Axonal tracing approaches have been extensively used in the domain of connectomics and they were found plausible in determining the large-scale connections of the mammalian cortex (=macroscale connectome). Emerging non-invasive neuroradiological approaches, like diffusion tensor MR imaging (DTI) and functional MRI (fmri) are promising candidates to acquire such data in humans. Here we sought to portray the possible applications of these techniques with particular attention on the mathematical frameworks that interpret macroscale connections as a complex network. The principle behind DTI and tractography techniques is that the possible (structural) pathways of information transfer can be traced by depicting the voxel-wise directionality and profile of water diffusion, which is hindered by axonal membranes. Consequently, the probability of existing structural connectivity can be determined between brain regions. On the contrary, functional MRI depicts the blood-oxygen level dependent contrast, which is presumably coupled with neuronal activity. Generally, this was feasible in revealing low frequency synchronisation between remote brain areas (=functional connectivity). In this decade, both in vivo determinants of connectivity were used in network-based interpretations such as graph theoretical analysis. We demonstrate our developments in this domain and forecast the possible role of graph analysis in understanding brain pathologies. Symposium 2. Unbiased strategies in drug discovery S2.1. Single cell transcriptome by sequencing all messages: reveals novel drug targets on neurons and adipocytes 1 James Eberwine, 2 Tamás Bártfai 10

11 1 University of Pennsylvania, USA; 2 The Scripps Research Institute, USA We report on an 'unbiased' molecular characterization of individual, adult neurons, active in a central, anterior hypothalamic neuronal circuit, by establishing cdna libraries from each individual, electrophysiologically identified warm sensitive neuron (WSN). The cdna libraries were analyzed by Affymetrix microarray. The presence and frequency of cdnas were confirmed and enhanced with Illumina sequencing of each single cell cdna library. cdnas encoding the GABA biosynthetic enzyme Gad1 and of adrenomedullin, galanin, prodynorphin, somatostatin, and tachykinin were found in the WSNs. The functional cellular and in vivo studies on dozens of the more than 500 neurotransmitters, hormone receptors and ion channels, whose cdna was identified and sequence confirmed, suggest little or no discrepancy between the transcriptional and functional data in WSNs; whenever agonists were available for a receptor whose cdna was identified, a functional response was found. Sequencing single neuron libraries permitted identification of rarely expressed receptors like the insulin receptor, adiponectin receptor 2 and of receptor heterodimers; information that is lost when pooling cells leads to dilution of signals and mixing signals. Despite the common electrophysiological phenotype and uniform Gad1 expression, WSN transcriptomes show heterogeneity, suggesting strong epigenetic influence on the transcriptome. Our study suggests that it is well-worth interrogating the cdna libraries of single neurons by sequencing and chipping. S2.2. Cytoskeletal changes in suicide subject's brain: is there a possibility of biomarkers for suicide prediction? Gábor Juhász, András Czurkó, Péter Gulyássy, Attila Simor, Katalin Adrienna Kékesi Laboratory of Proteomics, Eötvös Loránd University, Budapest, Hungary 11

12 Probing molecular brain mechanisms related to increased suicide risk is an important issue in biological psychiatry research. Gene expression studies on post mortem brains and the recently discovered ribosomal RNA gene promoter hyper-methylation indicate that there are extensive changes in protein expression prior to a successful suicide attempt; however, proteomic studies are scarce. Thus, we performed a DIGE proteomic analysis of post mortem tissue samples from the prefrontal cortex and amygdala of suicide victims to identify protein changes and biomarker candidates of suicide. We found 72 and 43 significant differences among our matched spots with the industry standard t test in the prefrontal cortex and amygdala, respectively. Because of the risk of false discoveries in these data we also made a more stringent statistical approach that reduced the number of significant spots to 39 and 9 respectively. From these we identified 67 proteins in the cortex and 7 proteins in the amygdala. These proteins are related to biological functions and structures such as metabolism, the redox system, the cytoskeleton, synaptic function, and proteolysis. Thirteen of these proteins (CBR1, DPYSL2, EFHD2, FKBP4, GFAP, GLUL, HSPA8, NEFL, NEFM, PGAM1, PRDX6, SELENBP1 and VIM,) have already been suggested to be biomarkers of psychiatric disorders at protein or genome level. From the statistically less stringent data pool we pointed out 11 proteins that changed in both the amygdala and the cortex, and from these, GFAP, INA, NEFL, NEFM, TUBA1 and VIM are interacting cytoskeletal proteins that have a functional connection to glutamate, GABA, and serotonin receptors. Moreover, ACTB, CTSD, GFAP and VIM displayed reverse changes that might be suitable for brain imaging studies. The reverse changes of ACTB, CTSD and GFAP in the two brain structures were validated by western blot analysis. S2.3. Development of novel drug targets in postpartum depression Árpád Dobolyi 12

13 Laboratory of Neuromorphology, Department of Anatomy, Histology and Embryology, Semmelweis University, Budapest, Hungary Postpartum depression occurring in 10-15% of births has the potential for significant impact on both the health of the mother and baby. Today s medications for depression target the brain s classical monoaminergic neurotransmitter systems and remain suboptimal, with many patients showing only partial remission or significant side-effects. Therefore, there is a large need to develop new therapeutic strategies designed to control postpartum depression. The relatively high ratio of mothers developing postpartum mood disorders suggests common mechanisms that could trigger the development of depression. Thus, proteins that selectively appear in the brain of mothers represent potential drug targets to treat postpartum depression. We describe tuberoinfundibular peptide of 39 residues expressed selectively in a posterior thalamic cell group in mother rats. It was demonstrated that this area conveys the suckling information towards different limbic and hypothalamic structures that in turn contain its receptor, the parathyroid hormone 2 receptor, a potential new drug target in postpartum depression. Another maternal neuropeptide, amylin, was identified in a microarray study by its selective expression in the maternal preoptic area, a region responsible for maternal motivation. The projection pattern of preoptic neurons in the brain overlaps with the distribution of amylin receptors, a potential drug target in postpartum depression. Changes in the maternal level of a number of other genes were also identified and confirmed by RT-PCR and in situ hybridization histochemistry. A cortical area involved in the etiology of depression, the medial prefrontal cortex was also examined by proteomics. Changes in protein levels were measured by 2-D gel electrophoresis followed by mass spectrometry identification of proteins. A number of genes and proteins whose expression change in the portpartum period were identified. Influencing the activity of some of maternally altered genes affects the behavior of animals in tests of anxiety and depression. Additional tests in mothers will provide targets for the development of new therapeutic strategies designed to control postpartum mood disorders. Support: OTKA K100319; Bolyai Fellowship of HAS. 13

14 S2.4. Molecular complexity and molecular networks: novel trends in psychiatric drug research György I Lévay Gedeon Richter Plc., Budapest, Hungary Symposium 3. Neurobiology of addiction S3.1. Physiological and anatomical properties of dopamine neurons Paul J. Bolam MRC Anatomical Neuropharmacology Unit, Department of Pharmacology, Oxford, United Kingdom Midbrain dopamine neurons are involved in a variety of functions and are particularly associated with reward mechanisms and addictive behaviour. In order to understand the contribution of dopamine neurons it is necessary to understand their properties and connections. The aim of this presentation to summarize some of the physiological and anatomical properties of dopamine neurons. Burst firing of dopamine neurons is associated with reward mechanisms and inhibition of firing in response to failure of rewards or aversive stimuli. Recordings from dopamine neurons in the substantia nigra pars compacta (SNc) of anaesthetized rats reveal a marked heterogeneity of responses to aversive stimuli (Brown et al 2009 J. Neuroscience 29: ). Light and electron microscopic analysis has 14

15 revealed the structural correlate of the inhibitory response. Inhibitory responses are correlated the with the relative extension of SNc neuron dendrites into the SN pars reticulata and the overall GABAergic innervation (Henny et al 2012 Nature Neuroscience 15: ). At the level of the striatum dopamine neurons provide a massive innervation. Dopaminergic axon terminals are in a position to modulate the flow of excitatory input derived from both the cortex and the thalamus although the quantitative electron microscopic analysis suggests that the innervation is non-specific (Moss & Bolam 2008 J. Neuroscience 28: ). Quantitative analyses suggest that individual SNc dopamine neurons give rise to thousand synapses at the level of the striatum. The massive axonal arbour is likely to put SNc dopamine neurons under a high energy demand and this may underlie their selective vulnerability in Parkinson s disease (Bolam & Pissadaki 2012 Movement Disorders 12: ; Pissadaki & Bolam 2013 Frontiers in Computational Neuroscience, in press). S3.2. Gene mapping opens up new avenues for treatment of addiction Csaba Vadász Laboratory of Neurobehavioral Genetics, New York University Medical Center, Nathan Kline Institute, 140 Old Orangeburg Road Alcohol dependence (alcoholism) and other addictions are complex diseases in which both genetic and environmental factors affect susceptibility. The consistently demonstrated 40-60% genetic variability in alcohol dependence and our profound ignorance of the underlying mechanisms which lead to addiction, highlight the need for identifying the genetic basis of susceptibility. The "genetic revolution" of the turn of the millennium raised hopes that the genetic underpinnings of complex neuropsychiatric disorders, including addictions, can be quickly identified. However, as recent reports of "lost heritability", and divergence of opinion 15

16 over the hidden variance indicate, it is now becoming clear that genetic complexity has been underestimated. New strategies take advantage of exciting progress in sequencing technologies, and integrate studies of human populations and model organisms. "Genetic noise-reduction" in animal models is an example of novel Quantitative Trait Locus (QTL) mapping strategies. Using a large panel of recombinant congenic mouse strains developed in our laboratory we mapped the QTL Ethyl alcohol consumption 2 (Eac2) to mouse chr. 6, and identified the underlying gene as cis-regulated glutamate receptor, metabotropic subtype 7 (Grm7; mglur7). The gene variant coding for lower mrna expression in the brain predisposes for higher alcohol consumption, which is consistent with studies on knockout mice showing increased voluntary alcohol drinking. Preliminary animal model and human genetic studies indicate translational potentials of targeting mglur7. Treatment with N, N'-dibenzyhydrylethane-1,2-diamine dihydrochloride (AMN082, 4 mg/kg, i.p.), a positive allosteric modulator of mglur7, reduced alcohol drinking, while AMN082 did not effect significantly consumption of sucrose which served as natural reward. mglur7 is the most conserved subtype among metabotropic glutamate receptors, and we hypothesized that its modulatory function has also been conserved in the course of evolution. Preliminary results of our collaborative human genetic studies show a trend of association between a GRM7 variant and an alcohol drinking phenotype developed on the basis of our animal model. In conclusion, recent developments in complex trait genetics suggest that integration of unbiased genome-wide animal model and human genetic strategies will be important in understanding addiction susceptibility, and development of new treatment avenues for addiction. S3.3. The ventral tegmental area in drug reward and beyond Christian Lüscher Department of Basic Neurosciences and Clinic of Neurology, University of Geneva, Switzerland 16

17 S3.4. Pathological gambling: wheel or wheelchair of Fortuna? Zoltán Janka University of Szeged, Department of Psychiatry, Szeged, Hungary Pathological gambling is a medically defined category and classified among impulse control disorders. However, it resembles in many aspects compulsion as uncontrollable urges appear to wager money to win (i.e. to rotate symbolically the wheel of Fortuna). Nevertheless, if gambling habit is under control in a socialized manner and does not extend to the compulsive field it can provide pleasure source for the risk-taking individual. Typical symptoms of pathological gambling are preoccupation with the topic, increasing the amount of money wagered, unsuccessful efforts to cut back/stop gambling behaviour and irritability/restlessness when doing that, alleviation of dysphoric mood, an effort to get even after losing, lies to conceal the habit, illegal acts to finance it, begging for loans from others, jeopardizing or destroying important relationships, job, career, and the behaviour is not due to manic mood elevation. Comorbidity with other psychiatric disorders is high, like alcohol and other substance abuse, depression/dysthymia, manic episode, generalized anxiety, panic, specific/social phobias, post-traumatic stress-disorder. Cognitive psychological investigations showed impairments in working memory, mental flexibility, planning, inhibition, and time management of the affected individuals. In vivo neuroimaging techniques revealed altered activation in specific brain areas (e.g. ventrolateral prefrontal cortex) in association with neurocognitive tests (Stroop, Iowa Gambling). Neurotransmitter systems (DA, 5HT, NA, opioidergic) have been shown to participate in gambling behaviour. Evidences for dysfunctional mesolimbic DA pathway, nucleus accumbens, aberrant salience attribution, deficits in motivational reward have been suggested in the pathophysiology. Serotonergic mechanisms have also been implicated, and further, therapeutic efforts with serotonin reuptake inhibitors point to the 17

18 involvement of 5HT. Molecular genetic investigations suggested some DA receptor gene allelic associations (DRD2, DRD3, DRD1) with pathological gambling. Treatment (psycho -and pharmacotherapy) is difficult as less than 10% of gamblers seek medical or psychological help and the attrition rate during therapy is high. Consequently, the outcome for the majority with this addictive behaviour is devastating: a complete existential collapse, i.e. starting to rotate the wheel of Fortuna leads directly and irresistibly to a handicapped state in the wheelchair of Fortuna. Symposium 4. Imaging techniques of the 21st century S4.1. Quantitative molecular differences in presynaptic active zones Zoltan Nusser Laboratory of Cellular Neurophysiology, Institute of Experimental Medicine, Hungarian Academy of Sciences, Budapest, Hungary Synapses of the central nervous system display remarkable structural and functional heterogeneities, the underlying mechanism of which is largely unknown. I will present our recent experiments obtained with two-photon imaging-based optical quantal analysis and correlated electron microscopic (EM) 3D reconstruction, showing that the probability of glutamate release (Pr) from local axon collaterals of hippocampal CA3 pyramidal cells scales linearly with the size of the active zone (AZ). Combined in vitro twophoton Ca2+ imaging from presynaptic boutons and correlated EM reconstruction of the imaged boutons revealed that the total fluxed Ca2+ following an action potential is also proportional with the AZ area. SDSdigested freeze-fracture replica immunogold labeling of the presynaptic 18

19 voltage-gated Ca2+ channel subunit Cav2.1 showed that this ion channel is confined to the AZ within the axon terminals. The density of Cav2.1 and that of other AZ proteins (e.g. Rim1/2) is uniform across AZs. The Pr and short-term plasticity patterns of axon terminals contacting parvalbumin or mglur1α immunopositive GABAergic interneurons also display great variability. I will also present some recent data demonstrating that the underlying mechanism might be a lower presynaptic Ca2+ channel density at the AZ of those terminals that synapse on mglur1α positive cells. Our results provide evidence for quantitative differences in the molecular composition of the presynaptic AZs of local axon terminals of CA3 pyramidal cells, with the consequence of allowing the fine tuning the release probability. S4.2. Fast two-photon in vivo and in vitro imaging in near-cubic-millimeter volume up to sub-millisecond temporal resolution 1 Balázs Rózsa, 1 Gergely Katona, 1 Gergely Szalay, 2 Pál Maák, 1 Kaszás Attila, 2 Máté Veress, 3 Dániel Hillier, 1 Balázs Chiovini, 3 Botond Roska 1 Two-Photon Imaging Center, IEM HAS, Budapest, Hungary; 2 Dept. of Atomic Physics, Budapest University of Technology and Economics, Budapest, Hungary; 3 Neural Circuit Laboratories, Friedrich Miescher Institute for Biomedical Resear, Basel, Hungary The understanding of brain computations requires methods that read out neural activity on different spatial and temporal scales. Following signal propagation and integration across a neuron and recording the concerted activity of hundreds of neurons pose distinct challenges, and the design of imaging systems has been mostly focused on tackling one of the two operations. We developed a high-resolution (PSF diameter in the center: 450 nm), fast (up to 55 microsecond temporal resolution), acousto-optic two-photon microscope with continuous three-dimensional trajectory and random-access scanning modes that reaches near-cubic-millimeter scan 19

20 range (over µm 3 ) and can be adapted to imaging different spatial scales. The system designed in ZEMAX and OSLO contains new design concepts: it physically separates the z-dimension focusing and lateral scanning functions to optimize the lateral AO scanning range; it allows the acoustic frequency chirps in the deflectors to be adjusted dynamically to compensate for astigmatism and optical errors; it involves a custom angular compensation unit to diminish off-axis angular dispersion introduced by the AO deflectors and it uses a high-na, widefield objective and high-bandwidth custom AO deflectors with large apertures. The resolution of the system allows simultaneous functional measurements in many fine neuronal processes, even in dendritic spines within a central core (~ μm 3 ) of the total scanned volume. Furthermore, the PSF size remained sufficiently low (PSFx < 1.9 μm, PSFz < 7.9 μm) to target individual neuronal somata in the whole scanning volume for simultaneous measurement of activity from hundreds of cells. With respect to the number of measurement locations on neuronal processes and cell bodies, we demonstrated fast 3D measurement of activity from 87 fine dendritic segments and over 500 cells included; however, the number of locations is not limited by the AO scanning method. The selection of active neurons in a volume that respond to a given stimulus was aided by the real-time data analysis and the 3D interactive visualization accelerated selection of regions of interest. S4.3. Diffusion tensor tractography in stereotactic functional brain surgery István Valálik Department of Neurosurgery, St. John s Hospital, Budapest, Hungary There are a significant number of patients with movement disorders, Tourette-syndrome (TS) and obsessive-compulsive disorder (OCD) who have not benefitted from pharmaceutical treatment or psychotherapy. Stereotactic radiofrequency thermo lesion and reversible and controllable 20

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